Abstract: For the past two decades, several labs including John York, Susan Wente, Steve Shears, Adolfo Saiardi and Solomon Synder have tried to elucidate how higher-order inositol phosphate 2nd messenger signaling molecules (inositols) regulate transcription, mainly examining single transcriptional units in yeast by genetic complementation/epistasis analyses. These studies focused on the completely conserved and ubiquitous inositol phosphate multikinase (IPMK, ipk2), as this kinase sits at the nexus of several pathways required for production of all higher inositols. IPMK activity is clearly required to rescue yeast phenotypes and transcripts from individual elements, but how inositols achieved this regulation was undescribed, as the chromatin effectors of inositols were unknown. In 2003, Erin O'Shea showed the kinase activity of IPMK regulates nucleosome sliding in yeast, Carl Wu and another group showed inositols regulate ATP-dependent chromatin remodelers Ino80 and Swi/Snf in vitro. However, inositol regulation of ATP-remodelers has not been built upon in any cellular studies since, despite availability of genomic approaches to examine open chromatin. We discovered a completely different way IPMK could regulate transcription, by directly phosphorylating a phospholipid while the lipid is bound in the hydrophobic cleft of a nuclear receptor. This model threatened to explain why the chromatin targets of IPMK were difficult to identify - they might be lipid- binding proteins, not inositol-binding proteins. This led us to attempt to identify other transcription factors regulated similarly by IPMK using genomics, presented in this proposal. In our human cell models we see IPMK is recruited to hundreds of transcriptional start sites, controlling transcript accumulation at those promoters in a kinase-dependent manner. But to our great surprise, GSEA immediately suggested IPMK primarily (but certainly not exclusively) regulates gene expression through histone deacetylases (HDACs). HDACs are transcriptional repressors shown in a series of structural biology papers by John Schwabe's group to require inositols, not lipids, for full activity in vitro. Indeed, histone acetylation increases upon IPMK loss, occurring at specific subsets of transcriptional start sites that recruit IPMK. All these aspects of IPMK functions in chromatin and at transcriptional start sites are novel. This proposal more deeply interrogates the new chromatin functions of IPMK described in our preliminary data, taking advantage of new chemical-genetics and other mutants of IPMK we have developed. Aim 1 identifies which of the new chromatin events are mediated most directly by IPMK, so mechanism can be studied. Aim 2 determines which IPMK-mediated chromatin events are shared between physiologically relevant model systems. Aim 3 resolves the mechanism of IPMK gene regulation. This proposal addresses long standing questions of how IPMK regulates gene expression while introducing a new chromatin-based 2nd messenger signaling paradigm that controls histone marks and transcription.